ABSTRACT Anti-depressant drug-induced liver injury is a major clinical concern, with up to 3% of patients treated showing evidence of liver damage. Duloxetine (DLX), the first-line and most prescribed antidepressant, carries a black box warning for its hepatotoxicity, but the mechanisms of DLX-induced liver injury remain largely unknown. Based on our preliminary data and clinical evidence, we hypothesize that DLX causes liver damage by disrupting hepatic lysophosphatidylcholine (LPC) homeostasis, and that suppression of DLX metabolism increases LPC accumulation and potentiates hepatotoxicity. The goal of this application is to determine the mechanism(s) of DLX-induced liver injury to enable the prediction and prevention of DLX hepatotoxicity. We will pursue this goal through three specific aims: (1) To determine if modifying LPC homeostasis impacts DLX toxicity, we will block LPC production pharmacologically with phospholipase A2 inhibitors and partially block LPC consumption genetically with Lpcat3-/- mice. We will determine DLX toxicity and assess LPC levels in the liver and blood. (2) To determine the mechanism by which DLX causes accumulation of LPCs, we will test whether DLX inhibits lysophospholipase, the enzyme that degrades LPC. We will test the mechanistic effect of DLX on lysophospholipase in vitro and in vivo using stable-isotope LPCs and known inhibitors. (3) We will use Cyp1a2-/-, Cyp2d-/-, or L-Porc/c mouse models to block CYP-mediated DLX metabolism and determine how this influences LPC levels and potentiates hepatotoxicity. We will use our novel human liver chimeric mice, in which mouse hepatic P450 oxidoreductase is non-functional, to assess Phase I metabolism of DLX and determine how inhibitors for specific human P450s impact hepatotoxicity of DLX. Completion of the proposed studies should help us understand the underlying mechanisms of DLX hepatotoxicity and provide an explanation for clinically observed DLX-drug interactions. Our research strategies could provide a general approach for the toxicological community to investigate drug-induced liver injury. These findings may lead to novel strategies for prediction and prevention of DLX hepatotoxicity in clinic and improvement of the DLX clinical safety profile.